P593: MULTIDIMENSIONAL ANALYSIS OF THE B CELL RECEPTOR OFFERS INSIGHT INTO THE ONTOGENETIC RELATIONSHIP OF MONOCLONAL B-CELL LYMPHOCYTOSIS WITH CHRONIC LYMPHOCYTIC LEUKEMIA
Authors: A. Agathangelidis, C. Galigalidou, A. Iatrou, L. Zaragoza-Infante, L. Scarfò, M. C. Maniou, P. Ranghetti, N. Pechlivanis, V. Junet, A. Skaftason, M. Tsagiopoulou, F. Psomopoulos, R. Rosenquist, P. Ghia, A. Chatzidimitriou, K. Stamatopoulos
Journal: HemaSphere (2022)

Background: The clonotypic B-cell receptor immunoglobulin (BcR IG) is key to the pathogenesis of CLL. Against that, however, scant information exists regarding the role of BcR IG in early stages of CLL ontogenesis, i.e. monoclonal B cell lymphocytosis (MBL). Aims: Here, we aimed to address this issue through the integrative analysis of: (i) the BcR IG gene repertoire, (ii) the transcriptome, and, (iii) the BcR IG reactivity profile, of both the high and the low-count MBL subtypes (HC-MBL and LC-MBL, respectively), complemented by comparisons to CLL. Methods: The study group comprised 17 individuals with LC-MBL and 14 with HC-MBL. “CLL-like” cells were flow-sorted. BcR IG NGS data was annotated with IMGT/HighV-QUEST and TRIPR. Clonotypes were defined as IG gene rearrangements with identical IG variable gene and CDR3 amino acid sequence. Stereotypy analysis in MBL cases focused on abundant clonotypes, which were compared against data from >39,000 CLL cases. Intraclonal diversification was assessed through graph networks of all distinct nucleotide variants of each clonotype. Transcriptome profiling was performed on polyA-selected RNA from 16 LC-MBL and 8 HC-MBL cases; comparisons were undertaken against RNAseq data from 74 CLL patients. Count normalization, differential gene expression and splice event analysis were performed with DESeq2, Limma and SGSseq, respectively; gene enrichment analysis was performed with Enrichr (p<0.05). BcR IG reactivity profiling was performed for 6 LC-MBL cases, 10 HC-MBL cases and 9 CLL cases against 123 autoantigens using a protein microarray; validation tests were performed with ELISA. Results: Clonality assessment revealed a monoclonal profile for most HC-MBL cases (86%), whereas LC-MBL cases were mainly oligoclonal (53%). Stereotypy analysis revealed a stronger immunogenetic connection of HC-MBL to CLL, since 19/47 abundant clonotypes (40%) from HC-MBL were assigned to CLL stereotyped subsets as opposed to only 19/167 (11%) from LC-MBL. Intraclonal diversification analysis was undertaken for: (i) 26 monoclonal cases (from HC-MBL or LC-MBL), (ii) 10 oligoclonal LC-MBL cases and 73 monoclonal CLL cases. Significant differences (p<0.05) were evident between oligoclonal MBL vs monoclonal MBL vs CLL; the former showed wide branching of variants due to distinct somatic hypermutations (SHM). In contrast, monoclonal MBL and CLL displayed progressively higher levels of variant connectivity due to the presence of shared SHMs, likely reflecting stronger antigenic pressure. Comparative transcriptomic analysis of either type of MBL vs CLL disclosed significant (p<0.05) down-regulation of genes in the Notch, AMPK and BcR signaling pathways. Moreover, LC-MBL showed overexpression of genes implicated in antigen processing and presentation and OXPHOS compared to either HC-MBL or CLL. In addition, both MBL types were characterized by virtual absence of alternative splicing, contrasting CLL where splicing events were identified in 14 genes of the BcR signaling pathway. Finally, BcR IG reactivity analysis revealed different patterns among individual cases, however all examined cases from MBL and CLL consistently bound a set of autoantigens, including myelin, centromere protein B, β2-microglobulin, C-reactive protein and the nucleosome. Summary/Conclusion: In conclusion, we highlight important differences between MBL and CLL stemming from the attributes of the BcR IG and the related signaling pathway. Overall, the reported findings allude to different maturation processes that likely contribute to shaping a distinct BcR IG signaling capacity in MBL, particularly LC-MBL, vs CLL.